Synthesis of dysideaproline E using organocatalysis.

(S)-4,4-Dichloro-3-methylbutanoic acid was prepared in 51% overall yield from commercially available starting materials using an organocatalytic transfer hydrogenation to 4,4-dichloro-3-methylbut-2-enal in the key step. The (S)-dichloro acid was used as an intermediate in the first total synthesis of dysideaproline E and a diastereomer confirming the structure of the natural product.

Synthesis of a series of phenylacetic acid 1-ß-O-acyl glucosides and comparison of their acyl migration and hydrolysis kinetics with the corresponding acyl glucuronides.

We report the synthesis of the 1-ß-O-acyl glucoside conjugates of phenylacetic acid (PAA), R- and S-α-methyl-PAA and α,α'-dimethyl-PAA, and measurement of their transacylation and hydrolysis reactivity by NMR methods. These are analogues of acyl glucuronides, the transacylation kinetics of which could be important in adverse drug effects. One aim of this work was to investigate whether, as previously postulated, the free carboxylate group of the acyl glucuronides plays a part in the mechanism of the internal acyl migration. In addition, such acyl glucosides are known to be endogenous biochemicals in their own right and investigation of their acyl migration propensities is novel. Our previously described selective acylation procedure has proved highly successful for 1-ß-O-acyl glucuronide synthesis and when subsequently applied to 6-O-trityl glucose, it gave good yields and excellent anomeric selectivity. Mild acidolysis of the O-trityl intermediates gave the desired acyl glucosides in excellent yield with essentially complete ß-selectivity. Measurement of the acyl glucoside transacylation kinetics by (1)H NMR spectroscopy, based simply on the disappearance of the 1-ß-isomer in aqueous buffer at pH 7.4, showed marked differences depending on the degree of methyl substitution. Further kinetic modelling of the isomerisation and hydrolysis reactions of the acyl glucosides showed considerable differences in kinetics for the various isomeric reactions. Reactions involving the -CH(2)OH group, presumably via a 6-membered ring ortho-ester intermediate, are facile and the α-glucoside anomers are significantly more reactive than their ß-counterparts. By comparison with degradation rates for the corresponding acyl glucuronides, it can be inferred that substitution of the carboxylate by -CH(2)OH in the acyl glucosides has a significant effect on acyl migration for those compounds, especially for rapidly transacylating molecules, and that thus the charged glucuronide carboxylate is a factor in the kinetics.

High-performance liquid chromatography/mass spectrometric and proton nuclear magnetic resonance spectroscopic studies of the transacylation and hydrolysis of the acyl glucuronides of a series of phenylacetic acids in buffer and human plasma.

The use of high-performance liquid chromatography/mass spectrometry (HPLC/MS) and proton nuclear magnetic resonance ((1)H NMR) spectroscopy for the kinetic analysis of acyl glucuronide (AG) isomerisation and hydrolysis of the 1-ß-O-acyl glucuronides (1-ß-O-AG) of phenylacetic acid, (R)- and (S)-α-methylphenylacetic acid and α,α-dimethylphenylacetic acid is described and compared. Each AG was incubated in both aqueous buffer, at pH 7.4, and control human plasma at 37°C. Aliquots of these incubations, taken throughout the reaction time-course, were analysed by HPLC/MS and (1)H NMR spectroscopy. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the calculated rates of reaction were much faster than for buffer and, in contrast to the observations in buffer, hydrolysis to the free aglycone was a significant contributor to the overall reaction.A diagnostic analytical methodology based on differential mass spectrometric fragmentation of 1-ß-O-AGs compared to the 2-, 3- and 4-positional isomers, which enables selective determination of the former, was confirmed and applied. These findings show that HPLC/MS offers a viable alternative to the more commonly used NMR spectroscopic approach for the determination of the transacylation and hydrolysis reactions of these AGs, with the major advantage of having the capability to do so in a complex biological matrix such as plasma.

Synthesis, transacylation kinetics and computational chemistry of a set of arylacetic acid 1beta-O-acyl glucuronides.

Many widely-used non-steroidal anti-inflammatory agents (NSAIDs), e.g. ibuprofen, are extensively metabolised as their acyl glucuronides (AGs), and the reactivity of these AGs raises important questions regarding drug safety and toxicity. In order to understand better the structure-reactivity of these metabolites, we have performed a detailed study of the synthesis, structural analysis and computed transacylation reactivity of a set of acyl glucuronides (AGs) of phenylacetic acids with varying alpha-substitution. A selective acylation procedure was used to prepare all the desired 1-(phenyl)acetyl-beta-D-glucopyranuronic acids 9, 12, 13 and 15 as single 1beta-anomers in good yields. Their reactivity was measured using 1H NMR spectroscopy in pH 7.4 buffer: the dominance of transacylation over hydrolysis in this system was confirmed together with the measurement of half-lives of the 1beta-isomers of the AGs. The half-lives ranged from 20 min for compound 9 to 23 h for 15. The lack of any significant concentration dependence of the reactivity suggests that the main mechanism is intramolecular. A novel computational chemistry and modelling study was performed on both the ground states of the AGs and the transition states for acyl migration to search for correlations with the kinetic data and to probe the mechanistic detail of the acyl transfer. An excellent degree of correlation was found between the calculated activation energies and the rates of transacylation. Especially, transition state analysis provided for the first time a firm mechanistic explanation for the slower kinetics of the (S)-isomer AG 13 compared to the (R)-isomer 12, thus throwing important light on the pharmacokinetic behaviour of marketed NSAIDs.

Identification of the thiophene ring of methapyrilene as a novel bioactivation-dependent hepatic toxicophore.

Methapyrilene (MP), a 2-thiophene H(1)-receptor antagonist, is a model toxicant in the genomic and proteomic analyses of hepatotoxicity. In rats, it causes an unusual periportal necrosis that is hypothetically attributed to chemically reactive and cytotoxic metabolites. We have characterized the bioactivation of MP by hepatic microsomes and primary rat hepatocytes, and we established a possible causal linkage with cytotoxicity. Methapyrilene tritiated at C-2 of the diaminoethane moiety ([3H]MP) was metabolized via an NADPH-dependent pathway to intermediates that combined irreversibly with microsomes (rat > mouse approximately human). This binding was attenuated by the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole and thiols but not by trapping agents for iminium ions and aldehydes. Reactive intermediates were trapped as thioether adducts of monooxygenated MP. Mass spectrometric and hydrogen/deuterium exchange analysis of the glutathione adduct produced by rat liver microsomes indicated that the metabolite was most probably a thioether of MP S-oxide substituted in the thiophene ring. The glutathione adduct was formed by rat hepatocytes and eliminated in bile by rats administered [3H]MP intravenously. MP produced concentration- and time-dependent cytotoxicity, depleted glutathione, and underwent irreversible binding to the hepatocytes before a significant increase in cell damage was observed. P450 inhibitors reduced turnover of the drug, production of the glutathione adduct, irreversible binding, and cytotoxicity but inhibited glutathione depletion selectively. MP underwent lesser turnover and bioactivation in mouse hepatocytes and was not cytotoxic. Analogs with phenyl and p-methoxyphenyl rings were much less hepatocytotoxic than MP. Hepatotoxicity in rats was diminished by predosing with 1-aminobenzotriazole. For the first time, a thiophene ring substituent is identified as a bioactivation-dependent toxicophore in hepatocytes.

Total synthesis of the marine natural product (-)-clavosolide A.

[Structure: see text] The total synthesis of the marine metabolite clavosolide A is reported which confirms the structure and absolute configuration of the natural product as the symmetrical diolide glycosylated by permethylated D-xylose moieties, 2.

Stereoselective synthesis of the tetrahydropyran core of polycarvernoside A.

[reaction: see text] A concise and stereoselective synthesis of the tetrasubstituted tetrahydropyran core of polycavernoside A was achieved in 55% overall yield from 3-benzyloxypropanal. A stereoselective allyl transfer reaction was used in the synthesis of enol ether 18 followed by a TFA-mediated cyclization to create the three new asymmetric centers in the tetrahydropyran with complete stereocontrol in a single-pot process.

Effective synthesis of 1beta-acyl glucuronides by selective acylation.

[reaction: see text] Acyl glucuronides are vital metabolites for many carboxylic acid containing drugs. We report an efficient new method for the chemical synthesis of these molecules by selective 1beta-acylation of allyl glucuronate with carboxylic acids catalyzed by HATU and then mild deprotection through treatment with Pd(PPh(3))(4) and morpholine. The method is effective for a range of aryl and alkyl carboxylic acids, including important drugs.

Total synthesis of a diastereomer of the marine natural product clavosolide A.

The first total synthesis of the reported structure of the sponge metabolite clavosolide A is described using a Prins cyclisation to assemble the tetrahydropyran core followed by manipulation of the side-chain, dimerisation and finally glycosidation.

Probing the mechanism of Prins cyclisations and application to the synthesis of 4-hydroxytetrahydropyrans.

Trapping intermediates on the Prins cyclisation pathway with carbon-based nucleophiles has given further insight into factors affecting the acid-mediated reactions of homoallylic alcohols with aldehydes, enabling the design of efficient syntheses of 4-hydroxy-2,6-disubstituted tetrahydropyrans.

Glycosidation with a disarmed glycosyl iodide: promotion and scope.

[reaction: see text] Glucuronyl iodide 1 has been studied in detail as a "disarmed" glycosyl donor. In a model reaction, using N-iodosuccinimide (NIS) as a promoter and 2-phenylethanol as acceptor, best results were obtained using NIS with I(2), followed by trimethylsilyltrifluoromethanesulfonate (TMSOTf). When a series of primary and secondary alcohols was glycosylated using these conditions, yields of 60-83% of beta-glucuronides were obtained. Various "nonheavy" metal salts also effectively catalyzed the model reaction but led to significant amounts of alpha-product with less reactive secondary alcohols.

Prins cyclizations: labeling studies and application to natural product synthesis.

The first syntheses of two natural products, catechols 1 and 2, isolated from Plectranthus sylvestris (labiatae), are reported. Oxygen-18 labeling studies support the proposed intermediacy of a stabilized benzylic cation in the acid-promoted cyclization of an aldehyde and benzylic homoallylic alcohol possessing an electron-rich aromatic ring. In contrast, with an electron-deficient aromatic ring the pathway via a benzylic cation is only minor. [reaction: see text]

Oxonia-cope rearrangement and side-chain exchange in the Prins cyclization.

[reaction: see text] Evidence is presented here for the mechanism of the Prins cyclization of benzylic homoallylic alcohols, which shows that the outcome of the reaction is dependent upon the substituents on the aromatic ring. The presence of an electron-rich aromatic ring favors an oxonia-Cope rearrangement yielding a symmetrical tetrahydropyran as the major product formed via a side-chain exchange process. In contrast, with electron-deficient aromatic rings the expected 2,4,6-trisubstituted tetrahydropyran is formed.

Stereoselective synthesis of 4-hydroxy-2,3,6-trisubstituted tetrahydropyrans.

[reaction: see text] Reaction of homoallylic alcohols with aldehydes in the presence of TFA gives, after hydrolysis of the ester, 4-hydroxy-2,3,6-trisubstituted tetrahydropyrans with the creation of three new stereocenters in a single-pot process. By varying the aldehyde component, a variety of functionalized side chains are installed at C-2. The utility of this approach is extended to the enantioselective synthesis of tetrahydropyrans with >99% ee.

Preparation, X-ray structure and reactivity of a stable glycosyl iodide.

Highly selective reaction of methyl tetra-O-pivaloyl-beta-D-glucopyranuronate 2 with iodotrimethylsilane or (Me3Si)2 and I2 affords, in excellent yield, the 'disarmed' glycosyl iodide 1 which has good stability at 20 degrees C and excellent stability at 0 degrees C; the X-ray crystal structure of 1 is described, along with a comparison of its utility as a glycosyl donor to that of the corresponding bromide.

NMR spectroscopic studies on the in vitro acyl glucuronide migration kinetics of Ibuprofen ((+/-)-(R,S)-2-(4-isobutylphenyl) propanoic acid), its metabolites, and analogues.

Carboxylic acid-containing drugs are often metabolized to 1-beta-O-acyl glucuronides (AGs). These can undergo an internal chemical rearrangement, and the resulting reactive positional isomers can bind to endogenous proteins, with clear potential for adverse effects. Additionally any 1-beta-O-acyl-glucuronidated phase I metabolite of the drug can also show this propensity, and investigation of the adverse effect potential of a drug also needs to consider such metabolites. Here the transacylation of the common drug ibuprofen and two of its metabolites is investigated in vitro. 1-beta-O-Acyl (S)-ibuprofen glucuronide was isolated from human urine and also synthesized by selective acylation. Urine was also used as a source of the (R)-ibuprofen, (S)-2-hydroxyibuprofen, and (S,S)-carboxyibuprofen AGs. The degradation rates (a combination of transacylation and hydrolysis) were measured using 1H NMR spectroscopy, and the measured decrease in the 1-beta anomer over time was used to derive half-lives for the glucuronides. The biosynthetic and chemically synthesized (S)-ibuprofen AGs had half-lives of 3.68 and 3.76 h, respectively. (R)-Ibuprofen AG had a half-life of 1.79 h, a value approximately half that of the (S)-diastereoisomer, consistent with results from other 2-aryl propionic acid drug AGs. The 2-hydroxyibuprofen and carboxyibuprofen AGs gave half-lives of 5.03 and 4.80 h, considerably longer than that of either of the parent drug glucuronides. In addition, two (S)-ibuprofen glucuronides were synthesized with the glucuronide carboxyl function esterified with either ethyl or allyl groups. The (S)-ibuprofen AG ethyl ester and (S)-ibuprofen AG allyl esters were determined to have half-lives of 7.24 and 9.35 h, respectively. In order to construct useful structure-reactivity relationships, it is necessary to evaluate transacylation and hydrolysis separately, and here it is shown that the (R)- and (S)-ibuprofen AGs have different transacylation properties. The implications of these findings are discussed in terms of structure-activity relationships.

Glucuronidation of steroidal alcohols using iodosugar and imidate donors.

We report a study of the glucuronidation of a number of important steroidal secondary alcohols. The alcohols studied are androsterone 7, epiandrosterone 8, 17-acetoxy-androstane-3alpha,17beta-diol 9, 11alpha-hydroxyprogesterone 10, and 3-benzoylestradiol 11. These were first glucuronidated using the Schmidt trichloroacetimidate method with variations in acyl substituent (viz. derivatives 2 and 3), Lewis acid catalyst and order of addition. The results are contrasted with those obtained using our recently described glycosyl iodide donor 4, catalysed either by N-iodosuccinimide (NIS) or various metal salts.